RESUMO
Development of primary liver cancer is a multistage process. Detailed understanding of sequential epigenetic alterations is largely missing. Here, we performed Infinium Human Methylation 450k BeadChips and RNA-Seq analyses for genome-wide methylome and transcriptome profiling of cirrhotic liver (n = 7), low- (n = 4) and high-grade (n = 9) dysplastic lesions, and early (n = 5) and progressed (n = 3) hepatocellular carcinomas (HCC) synchronously detected in 8 patients with HCC with chronic hepatitis B infection. Integrative analyses of epigenetically driven molecular changes were identified and validated in 2 independent cohorts comprising 887 HCCs. Mitochondrial DNA sequencing was further employed for clonality analyses, indicating multiclonal origin in the majority of investigated HCCs. Alterations in DNA methylation progressively increased from liver cirrhosis (CL) to dysplastic lesions and reached a maximum in early HCCs. Associated early alterations identified by Ingenuity Pathway Analysis (IPA) involved apoptosis, immune regulation, and stemness pathways, while late changes centered on cell survival, proliferation, and invasion. We further validated 23 putative epidrivers with concomitant expression changes and associated with overall survival. Functionally, Striatin 4 (STRN4) was demonstrated to be epigenetically regulated, and inhibition of STRN4 significantly suppressed tumorigenicity of HCC cell lines. Overall, application of integrative genomic analyses defines epigenetic driver alterations and provides promising targets for potentially novel therapeutic approaches.
Assuntos
Proteínas de Ligação a Calmodulina/genética , Carcinogênese/genética , Carcinoma Hepatocelular/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Hepatite B Crônica/complicações , Neoplasias Hepáticas/genética , Adulto , Idoso , Proteínas de Ligação a Calmodulina/biossíntese , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/metabolismo , Metilação de DNA , DNA de Neoplasias/genética , Feminino , Perfilação da Expressão Gênica , Hepatite B Crônica/genética , Hepatite B Crônica/patologia , Humanos , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
Accurate assessment of tumour heterogeneity is an important issue that influences prognosis and therapeutic decision in molecular pathology. Due to the shortage of protective histones and a limited DNA repair capacity, the mitochondrial (mt)-genome undergoes high variability during tumour development. Therefore, screening of mt-genome represents a useful molecular tool for assessing precise cell lineages and tracking tumour history. Here, we describe a highly specific and robust multiplex PCR-based ultra-deep sequencing technology for analysis of the whole mt-genome (wmt-seq) on low quality-DNA from formalin-fixed paraffin-embedded tissues. As a proof of concept, we applied the wmt-seq technology to characterize the clonal relationship of non-small cell lung cancer (NSCLC) specimens with multiple lesions (N = 43) that show either different histological subtypes (group I) or pulmonary adenosquamous carcinoma as striking examples of a mixed-histology tumour (group II). The application of wmt-seq demonstrated that most samples bear common mt-mutations in each lesion of an individual patient, indicating a single cell progeny and clonal relationship. Hereby we show the monoclonal origin of histologically heterogeneous NSCLC and demonstrate the evolutionary relation of NSCLC cases carrying heteroplasmic mt-variants.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Evolução Molecular , Heterogeneidade Genética , Genoma Mitocondrial , Neoplasias Pulmonares/genética , Biópsia , Evolução Clonal , Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Anotação de Sequência Molecular , MutaçãoRESUMO
Microsatellites are short repetitive sequences of two, three, or four bases, prone to base mispairing. Microsatellite instability (MSI) occurs frequently in various types of cancer due to a defective DNA mismatch repair system. Therefore, MSI analysis is an important tool in clinical research and molecular diagnostics. Mostly, polyacrylamide gel electrophoresis or capillary electrophoresis of labeled microsatellite sequences is used for the detection of MSI. Here we present a microfluidic-based electrophoresis technology for MSI analyses. Defined loci of microsatellites were PCR amplified and amplicons were analyzed by microfluidic-based electrophoresis. The electropherogram profiles of tumor and non-tumor derived DNA clearly revealed an individual pattern identifying differences in tumor-associated microsatellites. Detection of MSI by microfluidics turned out to be a simple and efficient procedure but less laborious than conventional approaches. Thus, the chip-based microfluidic electrophoresis is a simple, reliable, and robust technology for MSI detection, which allows label-free analyses of microsatellite amplicons within 30 min.
